Molecular and prognostic classification of advanced melanoma: a multi-marker microcontamination assay of peripheral blood stem cells

Abstract

The presence or absence of melanoma cells in human peripheral blood has recently been shown to be associated with disease prognosis, including overall survival. The detection of tyrosinase mRNA-positive circulating melanoma cells by reverse transcription-polymerase chain reaction (RT-PCR) has been limited to disseminated tumours expressing measurable amounts of this melanocyte-specific enzyme. To biologically classify both melanotic and amelanotic melanomas and to evaluate the clinical and prognostic relevance of tumour cell microcontamination, we examined autologous peripheral blood stem cell (PBSC) harvests from patients with advanced malignant melanoma prior to dose-escalated chemotherapy. To assay heterogeneous melanoma cell antigen expression, we developed a highly sensitive RT-PCR using four melanoma- and one tumour-associated antigen as molecular markers. Expression of the melanocyte-associated transcripts of tyrosinase, MART1/Melan-A, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) as well as the tumour-specific transcript of MAGE-3 was analysed by RT-PCR in PBSC harvests from 31 patients. Seven of the 31 PBSC harvests tested positive for one or more molecular markers: two patients for tyrosinase only, and one patient for MAGE-3 only, one patient for tyrosinase and MAGE-3, one for tyrosinase and MART1/Melan-A, and two patients for tyrosinase, MART1/Melan-A, TRP-2 and MAGE-3. mRNA-positive patients exhibited a significantly impaired overall survival (P = 0.0032), with a median survival of 3 months as opposed to 10 months in PBSC mRNA-negative patients. In conclusion, the use of this multiple-marker microcontamination assay allowed for molecular and prognostic classification of advanced malignant melanoma.

Molecular diagnostics in melanoma

“…Solutions to correct this presumed high falsenegative rate and more validly assess the clinical use of RT-PCR for CMC are to increase the volume of the blood sample, 257,258 use the more sensitive method of nested PCR, 266 and/or use multiple melanoma-specific biomarkers in RT-PCR assays. 214,255,293,298,[304][305][306] Tyrosinase expression may be more of a marker for risk of metastatic melanoma rather than a marker of tumor burden as the finding of CMC by RT-PCR gains clinical significance only when sequential determinations are performed during F/U and not all patients with positive results develop metastatic disease. 302 However, if an immunoassay to measure tyrosinase protein concentration in the serum is developed, such an assay could serve as an alternative to measuring S100-b protein because of its greater specificity.…”

Molecular markers of circulating melanoma cells

“…Tyrosinase‐related protein‐1 (TYRP‐1) and Tyrosinase‐related protein‐2/Dopachrome tautomerase (TYRP‐2/Dct) are involved in normal melanin synthesis. When included in a multi‐marker RT‐PCR assay using blood of early‐stage melanoma patients (Schrader et al., 2000), these genes showed limited value as independent indicators of melanoma progression. On the contrary, these markers had prognostic value in advanced stage melanomas with 89% of samples being DCT positive and significantly associated with tumour thickness (Takeuchi et al., 2004).…”

“…Although gp100 is highly expressed in melanoma tissues, it does not show significant correlation to disease progression (de Vries et al., 2001). Moreover, when included in a peripheral blood multi‐marker assay it was found to exhibit positive results in healthy donor blood samples (Keilholz et al., 2004; Schrader et al., 2000), questioning its specificity.…”

Biomarkers in melanoma: Stage III and IV disease

“…Tyrosinase RT-PCR III/IV + mRNA Shorter OS Marker of adjuvant treatment efficacy [36] Multimarker RT-PCR (tyrosinase, MART-1, TRP-1, MAGE-3) IV -mRNA + mRNA 10-month median OS 3-month median OS Undetectable circulating tumor cells possible marker of therapy effectiveness [67,68] S-100-α protein IV Changing levels Response to therapy [63] MIA IV IV…”